Introduction

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Persistent activation of opioid receptors results in the gradual loss of the pharmacological action of opiate compounds (homologous desensitization) and, in some cases, other compounds selective for distinctly different receptor systems (heterologous desensitization). Because opioid receptors are coupled to a variety of G proteins, and thereby regulate multiple signaling pathways, the biochemical mechanisms resulting in desensitization are complex and not fully understood. Multiple opioid-activated pathways have been implicated in this process, including activation of the phospholipase C-protein kinase C (PKC) pathway (Spencer et al., 1997; Strassheim et al., 1998). Desensitization probably also involves a complex array of interacting signaling pathways, including G protein receptor kinases, which may phosphorylate agonist-occupied receptors, and arrestins, which functionally uncouple the phosphorylated receptor from the G protein (Inglese et al., 1993; Chuang et al., 1996; Ferguson et al., 1996). To describe this phenomenon at the cellular level, the goal of this study was to examine the characteristics of desensitization resulting from short-term and prolonged opioid exposure in primary sensory neurons and to identify a potential target by which the loss of opioid response occurs.

An important signaling pathway regulated by µ-opioids involves the rapid inhibition of high voltage-activated Ca²⁺ channels (Wilding et al., 1995). In sensory neurons, N- and P/Q-type Ca²⁺ channels are affected predominantly, with lesser effects on other subtypes (Schroeder et al., 1991; Seward et al., 1991; Moises et al., 1994a; Rusin and Moises, 1995). Regulation of voltage-gated Ca²⁺ channels can have significant consequences on neuronal activity because they play a central role in membrane excitability and neurotransmitter release (Lipscombe et al., 1989; Luebke et al., 1993). Inhibition of Ca²⁺ channels by µ-opioids occurs through the receptor-mediated activation of a pertussis toxin-sensitive G protein (G_o; Moises et al., 1994b), resulting in a shift in the voltage-dependence of ion channel activation (Hille, 1994). Qualitatively, the partial reversal of the current inhibition and restoration of fast activation kinetics reflect, in part, this change in calcium channel gating when the cell is strongly depolarized. Voltage-dependent modulation of calcium channels has been shown to result from receptor-mediated dissociation of the G protein and subsequent binding of the -subunit to the calcium channel (Herlitze et al., 1996; Ikeda, 1996; Delmas et al., 1998a; Zamponi and Snutch, 1998). However, this does not account for all of the channel inhibition in sensory neurons.

This study was undertaken to investigate acute and long-term opioid densensitization with respect to Ca²⁺ channel regulation. These experiments confirm and extend the findings of Nomura et al. (1994). First, we examined whether the duration of exposure to the µ-opioid agonist [D-Ala²,N-MePhe⁴,Gly-ol⁵]-enkephalin (DAMGO) differentially affected G_o-mediated inhibition of high voltage-activated Ca²⁺ channels in neonatal rat dorsal root ganglion (DRG) neurons. Furthermore, it has been well documented that G_o-linked receptors inhibit voltage-activated Ca²⁺ channels through distinct voltage-sensitive and voltage-insensitive mechanisms (for review, see Jones and Elmslie, 1997). Because G_o activation inhibits different calcium channel types by different mechanisms, it was of interest to test whether the changes that occur with short-term exposure to DAMGO were selective for a specific channel type and whether this involved a voltage-dependent or -independent pathway.

	Materials and Methods

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Preparation of Neuronal Cultures

DRG neurons were prepared from 7- to 9-day-old Sprague-Dawley rats (Harlan Sprague-Dawley, Inc., Indianapolis, IN). All animal use procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the University of Rochester Committee on Animal Resources. The ganglia were dissected away from the lumbo-sacral region of the spinal cord and collected on ice in Dulbecco's PBS without Ca²⁺ or Mg²⁺ (Life Technologies, Grand Island, NY). After multiple rinses with the Ca²⁺/Mg²⁺-free medium, the ganglia were treated with trypsin (1.25 mg/ml; Life Technologies) and collagenase (3 mg/ml; Sigma Chemical Co., St. Louis, MO) for 30 min at 37°C. Enzymatic digestion was terminated by rinsing the cells in Hanks' buffered saline solution (Life Technologies) containing equine serum (10%; Hyclone Laboratories, Logan, UT) and DNase I (0.05 mg/ml; Worthington Biochemical Corporation, Lakewood, NJ). The cells were suspended in plating medium [minimum essential medium (Life Technologies) supplemented with nerve growth factor (50 ng/ml; Life Technologies), equine serum (5%; Hyclone Laboratories), fetal bovine serum (5%; Hyclone Laboratories), glucose (5 mg/ml; Sigma Chemical Co.), L-glutamine (2 mM; Life Technologies), penicillin (100 U/ml; Life Technologies), streptomycin (100 µg/ml; Life Technologies), and gentamicin (0.05 mg/ml; Life Technologies)] and mechanically dispersed by trituration. The suspension (100 µl/dish = 1/2 DRG/dish) was then plated on 35-mm culture dishes with laminin (Collaborative Biomedical Research, Bedford, MA) as the substrate. After a 2-h incubation at 37°C the volume was brought to 2 ml with 50% plating medium and 50% feeding medium [minimum essential medium containing equine serum (10%), nerve growth factor (50 ng/ml), glucose (5 mg/ml), and L-glutamine (2 mM)]. After 24 h, a 50% exchange with feeding medium was performed. The cultures were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO₂ with a 50% exchange of feeding medium performed once weekly. To maximize the use of all DRG neurons from each preparation and to conduct all experiments in cells that had been maintained in culture for 4 to 14 days without the use of compounds needed to inhibit the growth of nonneuronal cells, some DRG neurons from each preparation (before plating) were placed in plating medium containing 10% dimethyl sulfoxide (Sigma Chemical Co.) and stored at 80°C. When needed, these cells were warmed at 37°C for 2 min, plated (1/2 DRG/dish) and, after a 1-h incubation at 37°C, rinsed with plating medium. The medium was replaced with feeding medium and the cultures maintained as described above. Only neurons without processes and with adequate space-clamp (rapid deactivation of tail currents) were accepted for analysis. Freshly prepared neurons and those previously frozen were identical in their electrophysiological and pharmacological properties.

Electrophysiology

Whole-cell voltage-clamp recordings were executed with the whole-cell variation of the patch-clamp technique (Hamill et al., 1981). Glass recording patch pipettes were shaped from microhematocrit tubes (Chase Instruments Corporation, Norcross, GA) with a Sutter Instruments Flaming/Brown P-87 micropipette puller. Pipettes had resistances of 1.2 to 1.8 M when filled with the following recording solution: 140 mM CsMeSO₃, 10 mM HEPES, 5 mM EGTA [or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, tetracesium salt], 5 mM ATP-Mg²⁺, and 0.1 mM GTP-sodium [all reagents from Sigma Chemical Co., except 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; Molecular Probes, Eugene, OR]. The pH was adjusted to 7.35 with 1 N CsOH after the addition of ATP. The osmolarity was 5 to 10% less than the bath solution (310-320 mOsm). Aliquots of the internal recording solution were stored at 80°C and kept on ice after thawing. The cells were bathed in an external Ca²⁺ buffer (pH 7.35; 330-340 mOsm) containing 67 mM choline chloride, 5.3 mM KCl, 100 mM tetraethylammonium chloride, 5.6 mM glucose, 10 mM HEPES, 0.8 mM MgCl₂, and 5 mM CaCl₂ (all reagents from Sigma Chemical Co.).

Recordings were made at room temperature with an Axopatch 1-B patch-clamp amplifier (Axon Instruments, Foster City, CA). Pipette and whole-cell capacitance and series resistance were corrected by compensation circuitry on the amplifier. Series resistance compensation of 80 to 90% was possible without significant noise or oscillation. Whole-cell Ca²⁺ currents were evoked every 30 s by 100-ms voltage steps to +10 mV (test pulse) from holding potential (V_h) = 80 mV. In experiments in which the voltage-sensitive component of agonist regulation of Ca²⁺ channels was assessed, a 95-ms depolarizing prepulse to +100 mV was elicited 5 ms before the test pulse. Currents were filtered with a Bessel filter at 5 kHz (3 dB) and the records digitized at 5 kHz. Data acquisition and analysis was performed with pCLAMP software (versions 6.0 and 7.0; Axon Instruments) installed on a microcomputer with Pentium processor. Leak current was determined by a P/P6 protocol. This current was digitally subtracted from the relevant inward current to obtain the calcium current.

Solution Preparation and Application

DAMGO, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH₂ (CTOP; both from Research Biochemicals International, Natick, MA), and -conotoxin GVIA (Peptides International, Louisville, KY) were prepared as 5 mM, 625 µM, and 100 µM stock solutions in distilled water, respectively, partitioned into 20-µl aliquots, lyophilized, and stored at 20°C. On the day of the experiment, the lyophilized compound was dissolved in the external Ca²⁺ buffer at the desired concentration. BSA (0.1%; Sigma Chemical Co.) was included in the CTOP solution to minimize peptide binding to the application system. A 10-µl aliquot of DAMGO, reconstituted in distilled water, was added directly into the culture medium to achieve a final concentration of 5 µM for experiments in which neurons were exposed to the µ-opioid agonist for 24 h. Baclofen and naloxone hydrochloride (both from Sigma Chemical Co.) were prepared on the day of the experiment at the desired concentration in external Ca²⁺ buffer.

DAMGO, CTOP, baclofen, and naloxone were applied with a gravity-fed U-tube microperfusion system in which the microenvironment of the cell was continuously perfused with either external Ca²⁺ buffer (control) or drug-containing solution. Whole-cell Ca²⁺ currents were evoked 20 s after switching solutions. -Conotoxin GVIA was applied to the cell under study from a blunt-tipped (12-15-µm tip i.d.) glass micropipette positioned ~30 µm from the cell with pressure ejection (6-10 kilopascals) for a duration of 20 s. The puffer pipette was removed from the bath when not in use. In all experiments, drug concentrations were minimized in the remainder of the culture dish by the continuous gravity-fed influx (~0.3 ml/min) and vacuum-removed efflux of external bath solution. However, where long applications of drug were used (principally short-term desensitization experiments), only 1 cell/dish was used to eliminate prior exposure to agonist, antagonist, or toxin as a confounding influence on the measured response. Generally, experiments in which the effect of prolonged DAMGO exposure on Ca²⁺ channel regulation was tested, more than 1 cell/dish was studied.

Pretreatment and Testing Protocols

Prolonged DAMGO Exposure. DRG neurons were either left untreated or exposed to a 5 µM concentration of DAMGO for 24 h. After the pretreatment period, the culture medium (± DAMGO) was removed by rinsing the cells two to three times with external Ca²⁺ buffer. Healthy neurons were identified, the whole-cell recording was established, and voltage-activated Ca²⁺ currents were elicited. DAMGO (3 µM) or baclofen (50 µM) responses were assessed with the U-tube microperfusion application system.

Short-Term DAMGO Desensitization. After removal and exchange of the culture medium by rinsing two to three times and replacing with external Ca²⁺ buffer, whole-cell recordings in DRG neurons were initiated and voltage-activated Ca²⁺ currents elicited. Once the magnitude of the peak current had stabilized, DAMGO (3 µM) was continuously applied with the U-tube microperfusion application system. Calcium currents were elicited every 30 s and desensitization assessed by monitoring the increase in the peak current magnitude with continuous exposure to the µ-opioid agonist. On completion of desensitization (increase in peak current magnitude had stopped), drug application was terminated by switching the perfusion solution back to external Ca²⁺ buffer. Calcium currents were then reassessed in the absence of drug to determine the extent of current "rundown".

-Aminobutyric Acid (GABA)_B Modulation after Short-Term DAMGO Desensitization. Because evidence from pilot experiments suggested that a single, brief application of baclofen resulted in a reduced response on subsequent exposure, the protocol used to establish short-term DAMGO desensitization was modified from that described above to avoid the potential confounding influence of repeated baclofen exposure. After identification of a healthy DRG neuron and before establishing the whole-cell patch configuration, a 10-min DAMGO (3 µM) application was accomplished with the U-tube microperfusion system. The whole-cell patch configuration was then established and Ca²⁺ currents were elicited every 30 s until the peak current magnitude was stable (at this point we assumed, based on previous results, that both DAMGO desensitization and current stabilization were complete). Application of the DAMGO-containing solution was then stopped, the DAMGO removed by vacuum-removed efflux, and the perfusion solution switched to one containing only the external Ca²⁺ buffer. Calcium currents were again elicited every 30 s until the peak current magnitude was stable (i.e., "recovery" from the DAMGO-mediated inhibition was complete). The effect of a brief application of baclofen (50 µM ± conditioning prepulse) on peak current magnitude was then assessed.

Data Analysis

The magnitude and time of the whole-cell peak calcium current was determined with the peak detect feature of the pCLAMP software (Axon Instruments). With few exceptions, currents were elicited every 30 s. Cells exhibiting >7.5% rundown over the course of the experiment were excluded from the analysis. Analysis consisted of comparisons of current peak magnitude, time-to-peak, and percentage change in the peak current exhibited in the presence of drug. The magnitude of the change in peak whole-cell calcium current was defined as the difference between the current magnitude in the absence and presence of drug and expressed as a percentage of the control current magnitude (% change = 100 × [peak control current/peak current in presence of drug)/peak control current]). The frequency of µ-opioid responding cells also was determined. Drug-sensitive cells were defined as those exhibiting an inhibition of peak current magnitude of 10% in the presence of the agonist compared with control. The magnitude of desensitization was defined as the proportion of the initial drug response that was lost on completion of the desensitization paradigm. The voltage-dependent component of agonist-mediated inhibition was assessed with a two-pulse protocol. A test pulse to +10 mV was preceded by a 95-ms depolarizing prepulse to +100 mV with an interpulse interval of 5 ms. The current generated by this test pulse (P2) was compared with the test pulse (P1) that immediately preceded the two-pulse protocol. Reversal of agonist-mediated inhibition was quantified by determining the degree of current facilitation (facilitation ratio) after the conditioning prepulse and expressed as the ratio of the peak current amplitude after prepulse to the peak current without prepulse (P2/P1). Current inhibitions in the absence and presence of a conditioning prepulse were compared and expressed as the inhibition ratio (IR). The voltage-dependent component of current inhibition (percentage) was then estimated to be 100  100/IR. Means between different samples were compared with Student's two-tailed t test. Changes within cells were statistically compared with the paired Student's t test. Proportions were compared with the ² test. Except where indicated, data are presented as the mean ± S.E.

	Results

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µ-Opioid, but not GABA_B, Regulation of Voltage-Gated Ca²⁺ Channels Is Lost after Prolonged Exposure to DAMGO. Neonatal rat DRG neurons exhibited rapidly activating inward Ca²⁺ currents when elicited by a 100-ms depolarizing test pulse to +10 mV from a V_h of 80 mV. Of the 165 untreated DRG neurons tested with the µ-opioid selective agonist DAMGO (3 µM), 66% exhibited a 10% reduction in peak Ca²⁺ current ("DAMGO-sensitive"). Brief application of DAMGO resulted in reversible inhibition of the peak whole-cell Ca²⁺ current, an action that was completely blocked by coapplication of the µ-opioid antagonist CTOP (3 µM). The magnitude of peak current inhibition varied greatly in DAMGO-sensitive cells (10-74%; Fig. 1A) with a mean of 23.8 ± 1.2% (±S.E.; n = 108). Associated with the DAMGO-mediated decrease in peak Ca²⁺ current magnitude was a slowing of the activation kinetics, as indicated by an increase in the time-to-peak current magnitude (see current traces in Fig. 1B). Before the brief application of DAMGO, the peak of the Ca²⁺ current was achieved 15.2 ± 0.4 ms after the start of the voltage step to +10 mV. In contrast, the time-to-peak was significantly increased in the presence of the µ-opioid agonist (19.9 ± 1.2 ms; P < .001; paired two-tailed t test).

View larger version (33K): [in this window] [in a new window]   Fig. 1.   Effect of prolonged exposure to DAMGO on µ-opioid and GABAB regulation of high voltage-activated Ca2+ channels. A and C illustrate the distribution of untreated and DAMGO-treated neurons as a function of the magnitude of agonist-mediated inhibition of peak Ca2+ current. The magnitude of inhibition is expressed as the percentage of reduction in peak current after a brief application of the agonist compared with the control current just before the acute drug application. A, significantly greater proportion of untreated cells exhibited a definable DAMGO response (10% reduction of the peak current) compared with those that had been pretreated with DAMGO for 24 h (2 = 80.1; P < .001; n = 165 untreated, 73 pretreated). C, in contrast, the proportion of DRG neurons that were baclofen sensitive was not significantly different between the two treatment groups (2 = 0.36; P = .55; n = 74 untreated, 29 pretreated). B and D, whole-cell Ca2+ currents were evoked by 100-ms voltage steps to +10 mV from Vh = 80 mV in the absence (control) or presence of 3 µM DAMGO (B) or 50 µM baclofen (D) in neurons that were either untreated or exposed to 5 µM DAMGO for 24 h.

µ-Opioid and GABA_B Regulation of Voltage-Gated Ca²⁺ Channels Is Reduced after Short-Term Exposure to DAMGO. Continuous application of 3 µM DAMGO resulted in a significant decrease in µ-opioid-mediated inhibition of voltage-sensitive Ca²⁺ currents (short-term desensitization). Figure 2, A and B illustrate the effect of short-term application of DAMGO on whole-cell Ca²⁺ currents compared with its initial response (compare traces 2 and 3) and the failure of the response to recover completely within 10 min (traces 1' and 2'). Initial exposure to DAMGO reduced the magnitude of the peak Ca²⁺ current 34.0 ± 1.7% of the control current amplitude. In comparison, the peak Ca²⁺ current was inhibited by only 16.4 ± 1.0% (P < .001; n = 32) at the end of continuous DAMGO application. Once short-term desensitization was complete, there was a 52.2 ± 1.3% loss of DAMGO-mediated inhibition of the Ca²⁺ current (Fig. 2C). In addition, short-term exposure to DAMGO significantly affected the agonist-induced slowing of channel activation. The time-to-peak current amplitude with initial drug administration was 19.5 ± 1.7 ms and was significantly slower compared with that of the predrug current (11.6 ± 0.4 ms; P < .001; paired two-tailed t test). After short-term desensitization, however, channel activation in the presence of the agonist was more rapid (time-to-peak = 13.4 ± 0.5 ms). Although the time-to-peak did not return to the predrug value, short-term DAMGO exposure significantly affected the ability of the µ-opioid agonist to slow activation of the channel. So as to avoid confusion between current rundown and loss of response, any cell that exhibited >7.5% current rundown from its predrug current magnitude was excluded from the analysis.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Effect of short-term exposure to DAMGO on µ-opioid regulation of high voltage-activated Ca2+ channels. A, whole-cell Ca2+ currents were evoked by 100-ms steps to +10 mV from Vh = 80 mV. The traces were recorded in a representative DRG neuron at the times indicated (after patch rupture) in B. C, mean (±S.E.) DAMGO-mediated inhibition of Ca2+ currents is significantly reduced after an ~10-min exposure to the agonist (late DAMGO) compared with the initial response (initial DAMGO). ***P < .001, two-tailed paired t test, n = 32. D, results (mean ± S.E.) from a subset of neurons illustrated in C in which recovery was tested 10 min after the removal of the agonist. *P < .05; NS; n = 5.

µ-Opioid Regulation of Non-N-Type Ca²⁺ Channels Is Not Affected with Short-Term Exposure to DAMGO. Regulation of N-type Ca²⁺ channels has both voltage-dependent and voltage-independent components, whereas modulation of non-N-type channels is principally voltage-independent (Luebke and Dunlap, 1994; Bourinet et al., 1996; Delmas et al., 1998a; Sun and Dale, 1998). We therefore wished to test whether desensitization differed according to the targeted channel type and its mode of regulation with respect to voltage. If this were the case, DAMGO-mediated desensitization might not occur after eliminating the N-type channel component of the whole-cell current.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   µ-Opioid regulation of non-N-type Ca2+ channels does not exhibit short-term DAMGO desensitization. A, superimposed whole-cell Ca2+ currents evoked by a 100-ms test pulse to +10 mV from Vh = 80 mV at the times (after patch rupture) indicated in B. Currents were evoked before (1, 2) and after (3, 4, 5) a 20-s application of 10 µM -conotoxin GVIA. DAMGO-mediated inhibition was first determined for all targeted channels (trace 2). After elimination of the N-type component of the whole-cell current (trace 3), the effect of DAMGO (3 µM) was assessed on the remaining current. The opioid response targets primarily N-type channels (compare [1-2] and [3-4] in A and B and  and  in C) in these cells. Naloxone was used to terminate the DAMGO response after -conotoxin GVIA pretreatment to confirm that the small effect of DAMGO was due to an opioid action. C, magnitude of DAMGO-mediated inhibition of peak Ca2+ current on all targeted Ca2+ channels () and on non-N-type Ca2+ channels () for both early DAMGO versus late DAMGO. Data are the mean ± S.E. (n = 10; NS; P = .20), two-tailed paired t test).

Short-Term DAMGO Desensitization Significantly Attenuates the Voltage-Sensitive Component of µ-Opioid Regulation of Ca²⁺ Channels. Inhibition of high-threshold Ca²⁺ currents by G protein-coupled receptor agonists results from a shift in the voltage dependence of gating of the Ca²⁺ channels effected by specific membrane-delimited pathways (Bean, 1989; Hille, 1994). This change from a "willing" to "reluctant" channel state likely depends on the interaction of G protein -subunits with Ca²⁺ channel subunits (Herlitze et al., 1996; Ikeda, 1996; Ford et al., 1998; García et al., 1998). DAMGO-mediated inhibition of Ca²⁺ currents in neonatal rat DRG neurons exhibited many of the qualitative features of voltage-dependent inhibition. In addition to the DAMGO-mediated slowing of activation kinetics already described, a strong depolarizing prepulse both partially reversed DAMGO-mediated inhibition (facilitation) and restored the fast activation kinetics. Because one of the features of DAMGO desensitization was a shift in the activation kinetics back toward one of more rapid activation, the hypothesis that it was the voltage-dependent component of µ-opioid regulation of Ca²⁺ channels that was significantly attenuated during desensitization was tested.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Short-term DAMGO desensitization reduces the voltage-dependent relief of µ-opioid-mediated inhibition that is elicited by a strong depolarizing prepulse. A, superimposed whole-cell Ca2+ currents evoked by a 100-ms test pulse to +10 mV from Vh = 80 mV without (P1) or with (P2) a 95-ms depolarizing prepulse (+100 mV) 5 ms preceding the test pulse. Responses after the initial application of 3 µM DAMGO (A1), with or without a conditioning prepulse, are compared with responses after short-term continuous exposure to DAMGO (A2). The protocol used to establish short-term DAMGO desensitization was like that shown in Fig. 2. B, facilitation (mean ± S.E.) of peak current magnitude in the presence of the agonist (P2/P1) on initial application of the µ-opioid agonist (acute) and after desensitization (desensitized). C, comparison of DAMGO-mediated inhibition (mean ± S.E.) before () and after () a strong depolarizing prepulse on initial agonist application and after desensitization. This is summarized as the IR (ratio of inhibition before the prepulse to that after the prepulse, mean ± S.E.). D, scatter plot demonstrating that the remaining DAMGO-mediated inhibition in desensitized neurons () lacks a significant voltage-dependent component (expressed as the IR) compared with initial agonist exposure (). An IR 1.00 (dotted line) indicates that the inhibition is entirely voltage-independent. ***P < .001; NS; n = 15.

Short-Term DAMGO Desensitization Significantly Attenuates the Voltage-Sensitive Component of GABA_B Regulation of Ca²⁺ Channels. To test whether short-term DAMGO desensitization affected the voltage-dependent component of GABA_B-mediated regulation of high voltage-activated Ca²⁺ channels without the potential confounding influence of repeated baclofen exposure, an alternate desensitization protocol was used (see above; see Materials and Methods).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Short-term DAMGO desensitization reduces the voltage-dependent relief of GABAB-mediated inhibition that is elicited by a strong depolarizing prepulse. A, superimposed whole-cell Ca2+ currents evoked by a 100-ms test pulse to +10 mV from Vh = 80 mV without (P1) or with (P2) a 95-ms depolarizing prepulse (+100 mV) 5 ms preceding the test pulse. Traces show baclofen responses with and without conditioning prepulses in control DRG neurons (A1), and in separate cells pre-exposed for 15 min to DAMGO (A2). In this experiment, a modified protocol from that shown in Fig. 2 was used to establish short-term DAMGO desensitization. Before establishing the whole-cell patch configuration, a 10-min DAMGO (3 µM) application was accomplished with the U-tube microperfusion system. At this time the cell was patched and Ca2+ currents were elicited every 30 s until the peak current magnitude was stable (~5 min from patch rupture). Application of the DAMGO-containing solution was then stopped, the perfusion solution switched to one containing only the external Ca2+ buffer, and the DAMGO removed by continuous gravity-fed influx and vacuum-removed efflux of bath solution. Calcium currents were again elicited every 30 s until the peak current magnitude was stable (the assumption was that "recovery" from the DAMGO-mediated inhibition was complete). The effect of a brief application of baclofen (50 µM ± conditioning prepulse) on peak current magnitude was then assessed. B, comparison of baclofen-mediated inhibition (mean ± S.E.) before () and after () a strong depolarizing prepulse in control neurons (n = 12) and in cells pre-exposed to DAMGO for 15 min (n = 7).  indicates the mean responses expressed as the IR (see text). **P < .01 (two-sample t test), ***P < .001 (paired t test).

	Discussion

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This report demonstrates that there are two apparent components of µ-opioid-induced desensitization in neonatal rat DRG neurons, depending on the duration of agonist exposure, and that each likely involves different mechanisms. Prolonged exposure (24 h) to DAMGO resulted in a near complete loss of Ca²⁺ channel regulation by the µ-opioid agonist but did not affect the baclofen-mediated inhibition of targeted Ca²⁺ channels. In contrast, the early phase of desensitization developed rapidly, was maximal within 10 to 15 min of continuous DAMGO exposure, and began to reverse within minutes after removal of the agonist. During this period, not only was inhibition of targeted Ca²⁺ channels by DAMGO reduced by approximately 50% but also regulation of high voltage-activated Ca²⁺ channels by the GABA_B agonist baclofen was significantly attenuated. Moreover, the voltage-dependent component of agonist-mediated inhibition was significantly reduced or entirely lost as a consequence of short-term DAMGO-induced desensitization. The differing features of the two components of µ-opioid desensitization suggest separate mechanisms, as yet incompletely described, leading to the development of these states.

Activation of µ-opioid receptors results in the inhibition of Ca²⁺ current amplitude in agonist-sensitive DRG neurons by way of a membrane-delimited pathway that involves coupling of the receptor to the channel through a G_o-type G protein. However, current inhibition is never complete, indicating that only a subset of the total channel population is targeted by the activated receptor. Previous studies have shown (Rusin and Moises, 1995), and our data confirm, that the majority of channels affected by µ-agonists are N-type (85-90% of targeted current), with lesser effects on non-N-type channels. Roughly two-thirds of the whole-cell current in rat neonatal DRG neurons is N type; however, only half of that, constituting approximately one-third of the cell Ca²⁺ current, is inhibited by DAMGO.

Regulation of Ca²⁺ channels by DAMGO in DRG neurons exhibits in part the characteristic properties associated with voltage-dependent modulation after G protein activation. First, the inhibition of current amplitude is accompanied by a slowing of activation kinetics. Second, a strong depolarizing prepulse before the test depolarization restores the fast activation kinetics and partially reverses the current inhibition. G protein -subunits have been shown to cause neurotransmitter-like changes in channel voltage-dependent properties (Herlitze et al., 1996; Ikeda, 1996), presumably through an interaction with channel subunits (Bourinet et al., 1996; Ford et al., 1998; Sun and Dale, 1998) that readily reverses after strong depolarizing stimuli. In contrast, prepulse protocols yielded no facilitation of control currents, or of those currents remaining after block of N-type currents with -conotoxin GVIA. Together, these findings support the idea that the voltage-dependent component of G protein-mediated inhibition of Ca²⁺ currents in sensory neurons is selective for -conotoxin GVIA-sensitive N-type channels (Bean, 1989; Hille, 1994; but also see Luebke and Dunlap, 1994; Sun and Dale, 1997; Dolphin, 1998). The channel inhibition that remains after a strong depolarizing prepulse is presumably either due to a voltage-independent component or one that is less voltage sensitive to our prepulse protocol. We thus consider it likely that DAMGO inhibits N-type channels primarily via -subunits. The voltage-insensitive component of N- and non-N-type (presumably P/Q) channel inhibition is either mediated by -subunits for which the interaction with Ca²⁺ channels is less sensitive to voltage, or, perhaps, by -subunits (Jones and Elmslie, 1997; Delmas et al., 1998b).

A novel aspect of our results, one not described in other desensitization paradigms, is that desensitization within a single cell varied according to the µ-opioid-dependent signaling pathway. Specifically, short-term DAMGO exposure eliminated or significantly attenuated the voltage-dependent component of µ-opioid regulation of Ca²⁺ channels. In contrast, control currents or DAMGO-reduced non-N-type Ca²⁺ currents showed no prepulse facilitation. We thus conclude that little tonic inhibition of Ca²⁺ channels by G proteins exists in our preparation and that DAMGO-induced reduction of non-N-type Ca²⁺ currents is mediated by voltage-insensitive G protein subunits. The corollary is that the voltage-sensitive -dependent pathway mediating Ca²⁺ current reduction is most susceptible to desensitization. Our results also show, however, that there is a component of N-type channel inhibition by DAMGO that does not reverse by depolarizing prepulses and that does desensitize. It is possible that there is a voltage-sensitive component of -mediated channel inhibition that we cannot fully assay with our prepulse protocol. However, we favor the alternative explanation that there are pathways of Ca²⁺ channel inhibition that are either non--dependent (or nonvoltage-sensitive -dependent), but which are still under the influence of the desensitization process. Additional experiments will be required to address this more fully.

Our results therefore support the view that opioid desensitization in DRG neurons occurs by at least two time-dependent processes and that the acute phase of desensitization is remarkable by virtue of the apparent existence of different "compartments" of desensitization. We hypothesize that long-term desensitization is mediated primarily by receptor-targeting desensitization processes, such as G protein receptor kinases (Inglese et al., 1993; Chuang et al., 1996; Ferguson et al., 1996). This idea is supported by the longer time course required for the development of desensitization, the "completeness" of the loss of opioid signaling, and that the desensitization is homologous with respect to GABA_B receptors. We further hypothesize that acute desensitization is primarily a process that targets signaling components downstream of the receptor, perhaps by influencing the interaction of -subunits with Ca²⁺ channels. In support of this idea is the finding that the voltage-dependent -pathway is completely desensitized and that opioid-induced desensitization also reduces signaling within a convergent pathway (GABA_B) that uses the same G protein subtype and that targets similar Ca²⁺ channel subtypes. One mechanism by which this may occur is by PKC-induced phosphorylation of the targeted Ca²⁺ channel. PKC has been shown to phosphorylate the 1 subunit of the Ca²⁺ at or near the region to which the G-subunit binds (Swartz, 1993; De Waard et al., 1997). The effect of this phosphorylation would be an attenuation of G protein-mediated inhibition of the calcium channel. Predictably, in an expression system (Bourinet et al., 1996; Zamponi et al., 1997), PKC activation reduced opioid responses, an effect seen by others (King et al., 1999) and us (Xie et al., 1999) in cultured sensory rat neurons. Furthermore, in an animal lacking phospholipase-3, and which therefore has a reduced ability to activate PKC, opioid responses are increased (Xie et al., 1999). The challenge for future experiments will be to determine unequivocally the biochemical pathways mediating neuronal acute and long-term desensitization and their targeted signaling components.